Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha
Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.
KeywordsRalstonia eutropha Biofuel Branched-chain alcohol Isobutanol 3-Methyl-1-butanol Branched-chain amino acid
The authors thank Dr. Jens K. Plassmeier and Mr. Daan Speth for the helpful discussions; Mr. John W. Quimby and Dr. Qiang Fei for the critical review of this manuscript; and Ms. Amanda Bernardi for the assistance with R. eutropha isobutanol tolerance growth experiments. We also thank Professors Alexander Steinbüchel and Dieter Jendrossek for the generous gifts of the R. eutropha ADH mutant strains and Professor James Liao for the generous gift of the kivD gene. This work is funded by the US Department of Energy, Advanced Research Projects Agency—Energy (ARPA-E). We thank our ARPA-E collaborators Dr. Mark Worden and Ms. Yangmu Liu for their helpful discussions and support throughout the course of this study.
- Baer SH, Blaschek HP, Smith TL (1987) Effect of butanol challenge and temperature on lipid composition and membrane fluidity of butanol-tolerant Clostridium acetobutylicum. Appl Environ Microbiol 53:2854–2861Google Scholar
- Brigham CJ, Sinskey AJ (2012) Applications of polyhydroxyalkanoates in the medical industry. Int J Biotechnol Wellness Ind 1:53–60Google Scholar
- Gollop N, Damri B, Chipman DM, Barak Z (1990) Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms. J Bacteriol 172:3444–3449Google Scholar
- Jendrossek D, Kruger N, Steinbüchel A (1990) Characterization of alcohol dehydrogenase genes of derepressible wild-type Alcaligenes eutrophus H16 and constitutive mutants. J Bacteriol 172:4844–4851Google Scholar
- Karr DB, Waters JK, Emerich DW (1983) Analysis of poly-β-hydroxybutyrate in Rhizobium japonicum Bacteroids by ion-exclusion high-pressure liquid chromatography and UV detection. Appl Environ Microbiol 46:1339–1344Google Scholar
- Lu J, Brigham CJ, Rha C, Sinskey AJ (2012) Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16. Appl Microbiol Biotechnol. doi: 10.1007/s00253-012-4115-z
- Minty JJ, Lesnefsky AA, Lin F, Chen Y, Zaroff TA, Veloso AB, Xie B, McConnell CA, Ward RJ, Schwartz DR, Rouillard JM, Gao Y, Gulari E, Lin XN (2011) Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli. Microb Cell Fact 10:18–56CrossRefGoogle Scholar
- Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Potter M, Schwartz E, Strittmatter A, Voβ I, Gottschalk G, Stinbüchel A, Friedrich B, Bowien B (2006) Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–1262CrossRefGoogle Scholar
- Sambrook J, Rusell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Slater S, Houmiel KL, Tran M, Mitsky TA, Taylor NB, Padgette SR, Gruys KJ (1998) Multiple β-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 180:1979–1987Google Scholar
- Vollherbst-Schneck K, Sands JA, Montenecourt BS (1984) Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 47:193–194Google Scholar
- Westerfield WW (1945) A colorimetric determination of blood acetoin. J Biol Chem 161:495–502Google Scholar